Throughout this application various publications are referenced by their reference number within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the sequence listing. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Parasympathetic regulation of the rate of heart contraction is exerted through the release of acetylcholine (ACh), which opens a K.sup.+ channel in the atrium and thus slows the rate of depolarization that leads to initiation of the action potential (1,2). The coupling between binding of ACh to a muscarinic receptor and opening of the K.sup.+ channel occurs via a pertussis toxin (PTX)-sensitive heterotrimeric G-protein, G.sub.k (3-5), probably belonging to the G.sub.i family (6,7). Activation of this G-protein-activated K.sup.+ channel by G.sub.k does not require cytoplasmic intermediates (reviewed in refs. 8,9). However, a long-standing controversy exists as to which G-protein subunit couples to the KG channel. Purified .beta..gamma. subunit complex (10,11) and .alpha. subunits of G.sub.i family (6,7,12) activate the KG channel in cell free, inside-out patches of atrial myocytes. Activation by the .alpha. subunits occurs at lower concentrations than that by .beta..gamma., but seems to be less efficient (13); the relative physiological importance of each pathway, as well as of possible involvement of the arachidonic acid pathway (14), is unclear.
A channel similar or identical to the ACh-operated KG can be activated in the atrium by adenosine (15), ATP (16), and epinephrine (17), probably also via a G-protein pathway. Furthermore, in nerve cells various 7-helix receptors such as serotonin 5HT1A, .delta.-opioid, GABA.sub.B, somatostatin, etc., couple to similar K.sup.+ channels, probably through direct activation by G-proteins (18-22). The similarity of the channels and of the signaling pathways in atrium and some nerve cell preparations was strengthened by the demonstration of the coupling of a neuronal 5HT1A receptor (5HT1A-R), transiently expressed in atrial myocytes, to the atrial KG (23).
By electrophysiological and pharmacological criteria, the atrial KGA channel belongs to a family of inward rectifiers that conduct K.sup.+ much better in the inward than the outward direction, are blocked by extracellular Na.sup.+, Cs.sup.+ and Ba.sup.2+, and are believed to possess a single-file pore with several permeant and blocking ion binding sites (24). Many inward rectifiers are not activated by transmitters or voltage but seem to be constitutively active. Inward rectification of the atrial KGA channel is due to block of K.sup.+ efflux by intracellular Me.sup.2 (25), but for some channels of this family inward rectification may not depend on Mg.sup.2+ block (26,27). The molecular structures of atrial and neuronal KGs are unknown. Inwardly rectifying K.sup.+ channels structurally similar to voltage-activated K.sup.+ channels have been cloned from plant cells (28,29). Recently, the primary structures of two mammalian inward rectifier channels have been elucidated by molecular cloning of their cDNAs via expression in Xenopus oocytes: an ATP-regulated K.sup.+ channel from kidney, ROMK1 (30), and an inward rectifier from a macrophage cell line, IRKI (31). Both appear to belong to a new superfamily of K.sup.+ channels, with only two transmembrane domains per subunit and a pore region homologous to that of K.sup.+, Ca.sup.2+ and Na.sup.+ voltage-dependent channels (see ref. 32). It has been hypothesized that the structure of G-protein activated inward rectifying K.sup.+ channels should be similar to that of ROMK1 and IRK1 (31). Cloning of the atrial KGA channel and its expression in a heterologous system would be of importance not only for testing this hypothesis, but also because it will allow an as yet unexplored molecular approach to investigation of the mechanisms of direct G-protein-ion channel coupling. As a first step to cloning of the atrial KGA channel we have expressed it in Xenopus oocyte injected with atrial RNA and characterized the macroscopic current properties, including a preliminary characterization of G-protein coupling. We cloned the atrial KGA from a cDNA library derived from mRNA extracted from the heart of a 19 day old rat.